Reversed Phase Chromatography is a polarity-based separation method widely used in analytical and preparative HPLC. In this technique, a nonpolar stationary phase interacts with analytes dissolved in a polar mobile phase. Compounds are retained according to their hydrophobicity — the more hydrophobic the molecule, the longer it remains on the column.
Thanks to its broad selectivity range, high chemical stability, and compatibility with aqueous–organic eluents, RP-Chromatography has become the standard approach for applications in pharmaceutical analysis, biotechnology, environmental testing, and food chemistry. By adjusting the mobile phase composition or gradient, users can fine-tune selectivity and achieve reproducible separations across different systems and scales.
Modern reversed phase materials — such as C18, C8, C4, or Phenyl-bonded silica gels — provide optimized performance for small molecules, peptides, proteins, and other hydrophobic analytes, making RP-Chromatography the most flexible mode of liquid chromatography available.
Reversed Phase Chromatography represents the core of modern HPLC and UHPLC techniques. Its broad applicability, reproducibility, and compatibility with diverse detection systems (UV, MS, fluorescence) make it the preferred choice for both research and routine analysis. Whether for quality control, method development, or preparative purification, RPC offers reliable and scalable separation performance.
The stationary phase consists of porous silica particles modified with hydrophobic ligands such as C18, C8, or C4 chains. These bonded groups create a nonpolar surface that interacts with the hydrophobic regions of analyte molecules. Stationary phase selection — including ligand type, carbon load, pore size, and surface area — allows precise optimization for target molecule classes like small organic compounds, oligonucleotides, peptides, or proteins.
Typical mobile phases for RP-Chromatography are binary solvent mixtures of water with acetonitrile or methanol, often containing buffers or ion-pairing reagents to control pH and selectivity. Gradual changes in the organic solvent concentration (gradient elution) enhance separation efficiency and peak sharpness. The use of volatile buffers also ensures compatibility with LC-MS detection and preparative purification systems.
The retention mechanism in Reversed Phase Chromatography is dominated by hydrophobic interactions between the analyte and the stationary phase. Molecules with higher hydrophobicity exhibit stronger interactions and longer retention times. By adjusting mobile phase polarity, buffer strength, or temperature, chromatographers can fine-tune analyte retention and resolution, achieving optimal separation for both polar and nonpolar compounds.